Table I

Formulation of Treponema pallidum Culture Medium

Component Amount

1. Base medium

Earle's salt solution, lOx 100 ml

Essential amino acids (EAA),'1 50x 10 ml

Non-EAA," lOOx 10 ml

Vitamins," lOOx 10 ml

Glucose 2.5 g l-Glutamine, 200 mM 10 ml

Sodium pyruvate 100 mg

Dithiothreitol 150 mg FBS* 100-200 ml

2. Antioxidants

CoCl2 5 /xg

Cocarboxylase 2 fig

Mannitol 100 mg

Histidine 50 mg

Catalase 10,000 U

Superoxide dismutase 25,000 U

3. Ultrapure water to 1 liter

" Flow Laboratories (Rockville, MD). b Depends on the particular lot; some lots support growth better at 10% (v/v).

five is suitable for cultivation. All lots used in our experiments support growth that is 90% or greater than that of the reference lot.

Propagation of Treponema pallidum in Vivo and Harvesting Treponemes for in Vitro Cultivation. New Zealand White male rabbits (10-12 lb) are used for in vivo growth of T. pallidum and as a source of treponemes for inoculation of SflEp o tissue culture cells. For passage, 10 treponemes in 0.25 ml of TpCM are inoculated into rabbit testis. This inoculum originates from a 50% (v/v) glycerol frozen stock of treponemes (4 x 10s/ml) from a previous rabbit harvest. Ten days after infection, the rabbits are killed and the testes are aseptically removed. Treponemes are extracted from the testis in a laminar flow hood, using universal precautions. The testes are trimmed of any fatty tissue, minced in a sterile petri dish, and placed in a sterile 125-ml Erlenmeyer flask containing 12 ml of fresh TpCM base (see Table I). The flask is gassed with a mixture of 92% N2-5% C02-3% 02 (v/v) for 30 sec, sealed with a sterile silicone stopper, and placed on a orbital shaker operating at 120 oscillations per minute for 30 min. The TpCM base containing treponemes and testis extract is removed from the minced testicular tissue with a pipette and transferred into a 50-ml polypropylene conical centrifuge tube. The tube is briefly gassed with the gas mixture described above and sealed. The extract is then centrifuged at 500g for 10 min at 4° to remove gross debris. Dilutions of the supernatant (1:10 and 1:100) are prepared and the treponemes are counted by dark-field microscopy, using a Petroff-Hausser counting chamber. The remaining treponemal suspension is gassed and sealed for later use. This suspension is used as a source of inoculum and also for the preparation of testis extract.

In Vitro Cocultivation of Treponema pallidum with SflEp Cells

Cultivation of Tissue Culture Cells. The slow-growing SflEp cells are grown in Eagle's minimal essential medium (EMEM; GIBCO, Grand Island, NY) containing 10% (v/v) FBS unless otherwise noted. It is critical that the medium be free of any antibiotics because they will prevent infection of the monolayers with T. pallidum. Approximately 4 x 106 cells in 25 ml of EMEM are seeded into a 150-cm2 tissue culture flask. The medium is removed and replaced with fresh EMEM after 5 and 9 days of incubation. The cells are harvested and passaged every 2 weeks. The SflEp monolayers are usually set up at a confluency of 25% two days before infection with T. pallidum.

Preparation of Treponema pallidum Culture Medium for Cocultivation. TpCM is made from the base medium by adding the appropriate amounts of antioxidants listed in Table I and 3.33 ml of testis extract per 100 ml of TpCM. Testis extract is prepared by removing most of the treponemes from a portion of the treponemal suspension by centrifugation at 12,000g for 10 min. The supernatant is removed with a pipette and placed into a 50-ml polypropylene tube, gassed with the CO2-N2 mixture, and sealed. The extract is heat inactivated at 56° for 30 min. Any precipitate is removed by centrifugation at 12,000g for 10 min. The complete TpCM is alternately evacuated and gassed with a 5% CC>2-95% N2 mixture three times. The appropriate number of treponemes is added to the TpCM before infection of the tissue cultures.

Infection of SflEp Monolayers with Treponema pallidum. To infect tissue cultures, the EMEM is removed with a pipette and replaced with the appropriate volume of TpCM containing the treponemes. If the tissue culture vessel is a flask, it is briefly gassed with 5% C02 and 95% N2 and sealed. In contrast, tissue culture plates are placed in a vacuum chamber (Coy Laboratory Products, Ann Arbor, MI), evacuated (to -10 lb/in2), and gassed with a 5% CC>2/95% N2 mixture three times. When all the cultures have been prepared, the caps on tissue culture flasks are loosened and the tissue culture plates are removed from the vacuum chamber. All cultures are incubated at 34° in a Tri-Gas incubator (Forma Scientific, Marietta, OH), where a microaerophilic environment (3.5% 02, 5% C02, and 91.5% N2) is maintained throughout the cultivation period.

Quantitation of Treponemal Growth. Cultures are routinely counted after 5, 7, 10, 12, 14, and 17 days of incubation to obtain a complete growth curve. Treponemal growth is routinely monitored by harvesting the cultures with a mixture of 0.05% (w/v) trypsin and 0.53 mMEDTA in phosphate-buffered saline (PBS). To harvest the cultures, the TpCM is removed and placed into a 15-ml conical centrifuge tube. The cultures are then rinsed with 2 ml of PBS, which is removed and placed in the conical centrifuge tube with the TpCM. Three milliliters of trypsin-EDTA solution is added and the cultures are briefly gassed with 5% C02-95% N2. The centrifuge tubes are also gassed. After the cultures are incubated at 34° for 5 min, the culture monolayers are repeatedly triterated to dislodge all the tissue culture cells. This suspension is added to the TpCM in the conical centrifuge tube. If the sample is stored for more than 5 min before counting, it is gassed with CO2-N2. The concentration of treponemes in the TpCM suspension is determined by placing 10 ¡A in a Petroff-Hausser counting chamber followed by enumeration by dark-field microscopy.

Analysis of Effect of Atmospheric Oxygen Concentration on Growth of Treponema pallidum. To study the effects of oxygen tension on the growth of T. pallidum in coculture, multiple flasks of SplEp cells are infected with treponemes in TpCM lacking antioxidants. When the cocultures have been prepared, the tissue culture flasks are gassed with 5% CO2, 02 varying from 0.3 to 12.5%, and nitrogen. The flasks are then sealed and incubated at 34°. Treponemal growth is measured every 2 days over 16 days by harvesting and enumerating spirochetes from one or two flasks per oxygen concentration. By using this method we have found that treponemal growth in coculture occurs with 02 concentrations between 0.5 and 10%, with optimal growth between 1.5 and 5% (Fig. 1). Interestingly, this oxygen concentration (5%) is the same as that of many human tissues.5

Fig. 1. The effect of atmospheric oxygen concentration on the growth of T. pallidum cocultured with SflEp cells. Treponemes were grown in culture with SflEp cells in TpCM without antioxidants (base medium). The flasks were gassed with 5% CO2,02 varying from 0.3 to 12.5%, with the remainder nitrogen. Growth was monitored over 12 days and maximum growth was recorded.

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